The overall goal of this project is to develop a safe and broadly effective vaccine that will prevent group A streptococcal infections. Previous studies have shown that the surface M proteins, which are the major protective antigens, contain tissue-crossreactive epitopes as well as protective epitopes. The serotype-specific protective epitopes may be separated from potentially harmful autoimmune epitopes by using limited N-terminal peptides of M proteins. The protective M protein fragments representing multiple serotypes of group A streptococci may then be combined to form a multivalent vaccine. The specific aims of this proposal are: 1) To identify the primary structures of M proteins or other surface proteins that contain opsonic (protective) epitopes from serotypes of group A streptococci that are epidemiologically important and, therefore, necessary vaccine components, 2) To construct recombinant, multivalent vaccines that evoke optimal opsonic antibody responses in laboratory animals against 26 different serotypes of group A streptococci, 3) To test immune rabbit sera evoked by multivalent vaccines for opsonic and bactericidal antibodies against clinical isolates of group A streptococci collected from children with pharyngitis in 10 geographic sites in the U.S., 4) To develop strategies of intranasal delivery of multivalent M protein-based vaccines that result in secretory and systemic immune responses, and 5) To directly compare the protective immunogenicity of multivalent M protein-based vaccines delivered to mice via either the intramuscular or intranasal routes. In our preliminary studies, we have identified six epidemiologically important serotypes of group A streptococci that are not opsonized by antisera against the N-terminal M protein peptides. We propose a series of experiments to determine the covalent structures of the M proteins, M-like proteins, or other surface proteins that contain opsonic epitopes so that these M serotypes may be included in multivalent vaccines. We will construct a 26-valent vaccine composed of 4 different recombinant, hybrid proteins. The individual hybrid proteins will be tested for protective and tissue-crossreactive immunogenicity after intramuscular injection of rabbits. Because mucosal delivery of streptococcal vaccines may have both immunological and practical advantages over parenteral delivery, we will assess different strategies of intranasal delivery and then directly compare the protective efficacy of i.n. vs i.m. vaccines in mice. The studies should provide the detailed information needed to develop a safe and effective multivalent vaccine that could prevent the majority of streptococcal infections in North America and Western Europe.